We seek support in the form of a U19 Cooperative Agreement to characterize the microbicidal potential of RNA interference (RNAi) in preventing and treating infections caused by HIV-1, HSV and HPV. RNAi refers to post-transcriptional and post-translational gene silencing by single and double stranded RNA of approximately 19-21 nucleotide length. In this proposal, we will confine our studies to short ds RNA that have been termed short interfering RNA (siRNA). Upon cellular introduction, siRNA are incorporated into the RNAi Silencing Complex (RISC) and mediate the degradation of mRNA in a sequence-specific manner leading to potent inhibition of protein expression. Using animal models, we have demonstrated that siRNA can be introduced into vaginal mucosa as liposomal formulations. Detailed studies have revealed that a single vaginal siRNA treatment reduces levels of targeted proteins by up to 90% over a one-week period. We propose to identify siRNA to selectively degrade viral and/or host proteins implicated in the transmission and/or replication of HIV-1, HSV and HPV. After in vitro confirmation of siRNA gene silencing potency and anti-viral activity, animal models will be used to define the kinetics of targeted protein knockdown following vaginal siRNA treatment. While liposomal formulations will allow the immediate pursuit of these proof-of-principle studies, considerable effort will be devoted to nanocapsule formulation of siRNA to allow for efficient mucosal uptake and favorable toxicity profile. Simultaneous investigations will reveal whether chemical modifications to the siRNA structure itself increase the activity profile of the molecules. Thus, at the 2.5 year mark, we will have identified chemically optimized siRNA molecules that reduce the expression of validated pathogen-related targets in vivo after their vaginal administration as non-toxic nanocapsules. Thereafter, all effort will be directed to defining in vivo efficacy of siRNA in animal models of HIV-1, HSV and HPV transmission and infection. These goals will be pursued in four projects and two cores in a highly coordinated manner: Project 1: HIV-1 prevention (Ramratnam,Mansfield), Project 2: HSV prevention and treatment (Herold), Project 3: siRNA structural optimization (Alexander, Shaw), Project 4: HPV prevention and treatment (Lambert, Oz, Marx), Core A: Administrative (Ramratnam), Core B: Formulation (McDonough). At the end of the project, we will have generated pre-clinical data to justify the further evaluation of siRNA microbicides in human subjects.